Department of Pathology, University of Washington, Seattle, WA 98195, USA.
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Int J Mol Sci. 2019 Jun 8;20(11):2813. doi: 10.3390/ijms20112813.
Different phenotypes of normal cells might influence genetic profiles, epigenetic profiles, and tumorigenicities of their transformed derivatives. In this study, we investigate whether the whole mitochondrial genome of immortalized cells can be attributed to the different phenotypes (stem vs. non-stem) of their normal epithelial cell originators. To accurately determine mutations, we employed Duplex Sequencing, which exhibits the lowest error rates among currently-available DNA sequencing methods. Our results indicate that the vast majority of the observed mutations of the whole mitochondrial DNA occur at low-frequency (rare mutations). The most prevalent rare mutation types are C→T/G→A and A→G/T→C transitions. Frequencies and spectra of homoplasmic point mutations are virtually identical between stem cell-derived immortalized (SV1) cells and non-stem cell-derived immortalized (SV22) cells, verifying that both cell types were derived from the same woman. However, frequencies of rare point mutations are significantly lower in SV1 cells (5.79 × 10) than in SV22 cells (1.16 × 10). The significantly lower frequencies of rare mutations are aligned with a finding of longer average distances to adjacent mutations in SV1 cells than in SV22 cells. Additionally, the predicted pathogenicity for rare mutations in the mitochondrial tRNA genes tends to be lower (by 2.5-fold) in SV1 cells than in SV22 cells. While four known/confirmed pathogenic mt-tRNA mutations (m.5650 G>A, m.5521 G>A, m.5690 A>G, m.1630 A>G) were identified in SV22 cells, no such mutations were observed in SV1 cells. Our findings suggest that the immortalization of normal cells with stem cell features leads to decreased mitochondrial mutagenesis, particularly in RNA gene regions. The mutation spectra and mutations specific to stem cell-derived immortalized cells (vs. non-stem cell derived) have implications in characterizing the heterogeneity of tumors and understanding the role of mitochondrial mutations in the immortalization and transformation of human cells.
不同表型的正常细胞可能会影响其转化衍生物的遗传谱、表观遗传谱和致瘤性。在这项研究中，我们研究了永生化细胞的整个线粒体基因组是否可以归因于其正常上皮细胞起源的不同表型（干细胞与非干细胞）。为了准确确定突变，我们采用了双脱氧测序，它在目前可用的 DNA 测序方法中具有最低的错误率。我们的结果表明，整个线粒体 DNA 的绝大多数观察到的突变都发生在低频（罕见突变）。最常见的罕见突变类型是 C→T/G→A 和 A→G/T→C 转换。干细胞衍生的永生化（SV1）细胞和非干细胞衍生的永生化（SV22）细胞之间同质点突变的频率和谱几乎完全相同，这证实了两种细胞类型都来自同一个女人。然而，SV1 细胞中罕见点突变的频率（5.79×10）明显低于 SV22 细胞（1.16×10）。SV1 细胞中罕见突变的频率明显较低，与 SV1 细胞中相邻突变的平均距离比 SV22 细胞长的发现一致。此外，线粒体 tRNA 基因中罕见突变的预测致病性在 SV1 细胞中比在 SV22 细胞中低（低 2.5 倍）。虽然在 SV22 细胞中鉴定出了四个已知/确认的致病性 mt-tRNA 突变（m.5650 G>A、m.5521 G>A、m.5690 A>G、m.1630 A>G），但在 SV1 细胞中未观察到这些突变。我们的研究结果表明，具有干细胞特征的正常细胞的永生化导致线粒体突变减少，特别是在 RNA 基因区域。源自干细胞的永生化细胞（与非干细胞衍生）的突变谱和突变具有表征肿瘤异质性和理解线粒体突变在人类细胞永生化和转化中的作用的意义。
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